Method for generating saccharide fragments

ABSTRACT

A method for depolymerizing polysaccharides containing into saccharide fragments using ozonolysis is described.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This work was supported in part by National Institutes of Health grantsAI 30628, AI 75326, AI23339, and AI 25152. The government has certainrights in the invention.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.60/042,416 filed Mar. 26, 1997, which is incorporated herein in itsentirety.

BACKGROUND OF THE INVENTION

This invention is in the general field of methods of preparingsaccharide fragments.

Saccharides are important as commodity chemicals and are used often infood and industrial applications. They are also important specialtychemicals in biotechnology, e.g., in the preparation of antibiotics orantibodies, as antigens for vaccines, or as diagnostic reagents.

Saccharides may be obtained from natural sources or synthesizedenzymatically or chemically. Synthesis of saccharides having more thanabout five monosaccharide units often is difficult, especially if one ofthe units is sialic acid, which is acid labile. Enzymatic synthesis islimited by the available enzymes and substrates and may be relativelyexpensive.

While natural polysaccharides are sometimes available, in somesituations, their use presents problems when they are too large. Forexample, many food and industrial applications require polysaccharidesof specific sizes, and some native polysaccharides may be far too large.In some applications, decreasing the size of the polysaccharide canimprove ease of handling and lower production costs. Where across-linked saccharide is desired, e.g., to enhance immunogenicity whenused in a vaccine, available materials of high molecular weight may forminsoluble gels when cross-linked. Reducing the chain length of thestarting saccharide can avoid this problem.

Polysaccharides can be cleaved into smaller molecular weight fragmentsby acid, base, or enzymatic-catalyzed hydrolysis. Acid catalyzeddegradation may cleave polysaccharides nonselectively in bothcarbohydrate and other functional moieties, yielding inconsistentproducts or non-functional products. For example, sialic acids are foundon the carbohydrate moieties of many biologically importantpolysaccharides and can be determinants of biological functions,including recognition and attachment. They can also be determinants ofepitopes for antibody generation and as such should be conserved inattempts to generate saccharide fragments, from polysaccharides. Sialicacids, however, are readily removed by acid.

Enzymatic hydrolysis of polysaccharides can be highly specific but it isusually limited to applications where an enzyme with the desiredspecificity is readily available. Some saccharide fragments mayalternatively be isolated directly from natural sources, but thesenaturally occurring shorter polysaccharides typically exist in limitedquantity. In some cases, saccharide fragments may be chemically and/orenzymatically synthesized. However, even in those cases the enzymes andsubstrates necessary to conduct the synthesis may be expensive. Ingeneral, the synthesis of saccharide fragments of more than fivemonosaccharide residues can be extremely difficult.

SUMMARY OF THE INVENTION

The invention is based on the discovery that ozone can be used to cleavepolysaccharides to yield useful shorter-length saccharide fragments of adesired length, which generally retain structural features of thepolysaccharide.

Accordingly, the invention features a method for producing a saccharidefragment by oxidizing a larger polysaccharide having at least onecovalent bond between a C1 anomeric carbon of an aldose residue and anoxygen atom of a second monosaccharide residue in a β-D glycosidiclinkage. The method can also be used when the covalent bond is in theform of an α-L linkage. Typically, the β-D and α-L linkages will exhibitsimilar reactivities. Similarly, the β-D and α-L linkages will alsoexhibit similar reactivities. The method comprises protecting freehydroxyl groups on the larger polysaccharide; reacting the largerpolysaccharide with ozone to oxidize the C1 anomeric carbon, thusconverting the aldose residue into an aldonic acid ester residue; andcleaving the aldonic acid ester residue to form the saccharide fragment.

In another aspect, the invention features a method of preparing ansaccharide fragment by oxidizing a larger polysaccharide having at leastone covalent bond between a C1 anomeric carbon of an aldose residue andan oxygen atom of a second monosaccharide residue in an α or βglycosidic linkage (henceforth, referred to as the "one-step" method).

The glycosidic linkage can be in any form, e.g., α-L, α-D, β-L or β-D.As is mentioned above, the β-D and α-L linkages will typically exhibitsimilar reactivities, and the α-D and β-L linkages will exhibit similarreactivities.

The one-step method comprises reacting the larger polysaccharide withozone to cleave a bond linking two monosaccharide subunits in thepolysaccharide, resulting in the formation of the saccharide fragment.

The larger polysaccharide in the methods described herein can be fromany source and can contain labile residues, e.g., sialic acid.Preferably, the larger polysaccharide is substantially pure. The largerpolysaccharide is substantially purified when it is separated from thosecellular components which accompany it in its natural state. Similarly,the saccharide fragment may optionally be subsequently purified. By apurified saccharide fragment is meant a saccharide fragment separatedfrom the starting polysaccharide.

For purposes of diagnosis and vaccine development, the polysaccharidemay be from a bacterial pathogen, e.g., a group B Streptococcus capsularpolysaccharide, such as GBS type I, II, III, IV, V, VI, VII, and VIII;the O-antigen of a lipopolysaccharide; a capsular polysaccharide ofStaphylococcus aureus, e.g., the Staphylococcus aureus type 5 or type 8antigens; the capsular polysaccharide of Streptococcus pneumonia; andthe capsular polysaccharide of Bacteroides fragilis.

The ozone can be added in solution, generated in-situ, or be deliveredfrom an external source, e.g, bubbled in.

The aldonic acid ester intermediate can be cleaved by a nucleophile,e.g., a hydroxyl ion, an amine, a thiol, or a carbanion. The aldonicacid ester intermediate may alternatively be cleaved by heating orhydrolysis.

The invention also includes a method for producing antibodies usingsaccharide fragments produced by ozonolysis. The saccharide fragmentscan be conjugated to a carrier to create an immunogen, after which theimmunogen is injected into a suitable host. Any recognized host issuitable, e.g., rabbit, rat, mouse, goat. Either polyclonal ormonoclonal antibodies can be generated.

The invention has many advantages. The methods enable degradation of anypolysaccharide containing a glycosidic linkage. The one-step procedureallows ozonolysis to take place in an aqueous solution and without theneed for pretreating the starting polysaccharide. In addition, theone-step procedure can be used to depolymerize polysaccharidescontaining any glycosidic linkage.

If cleavage takes place at the same glycosyl residue, saccharidefragments with the same repeating unit structure can be recovered fromabundant, naturally occurring polysaccharides.

Saccharide fragments produced by this method can be easily modified andlinked to other molecules (e.g., protein carriers). This can make themuseful in drug and vaccine design. The saccharide fragments may also betagged with chromophores, biotins, peptides, and lipids and thus havediverse potential applications.

A still further advantage of the invention is that it is possible tovary the molecular weight of saccharide fragments generated by varyingthe ozonolysis conditions.

As used herein, "saccharide fragment" is any complex carbohydrate whichis formed according to the invention from a starting material which is a"starting polysaccharide". Thus, while the product is always smallerthan the starting material, no particular size limitation is implied oneither the starting material or the product. The size of the startingmaterial generally will be dictated by the source of polysaccharide thatis readily available. The size of the saccharide fragment will be afunction of various factors, such as the desire for a small moleculethat is more conveniently adapted to the end use (e.g., solubilized orreacted with labels), consistent with the need to conserve configurationor properties (e.g., immunological properties) of the larger startingmaterial. In general the polysaccharide starting material will have morethan 10 saccharide units, and it will be of a size dictated by itsnatural source and techniques for recovering it from that source. Theresulting saccharide fragment cleavage product will have more than 1unit, and typically will be smaller than 100 units. The saccharidefragments produced by the invention may in some cases be much longerthan 100 units. When the term "oligosaccharide" appears herein, it isunderstood that the term is synonymous with "saccharide fragment".

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the repeating unit structure of GBS (Group Bstreptococcal) capsular polysaccharides.

FIG. 2 is a graph showing the elution profile following liquidchromatography of saccharide fragments generated from type II GBSpolysaccharide after treatment with ozone.

FIG. 3A-D are the ¹ H NMR spectra of type II GBS native polysaccharideand the saccharides of peaks 3 to 1 of FIG. 2, respectively.

FIG. 4 is a graph showing the elution profile of saccharide fragmentsgenerated from type VIII GBS polysaccharide after treatment with ozonefor the indicated amounts of time.

FIG. 5 is the ¹ H NMR spectrum of pooled 7 Kda saccharide fragmentsgenerated from type VIII GBS polysaccharide after treatment with ozone.

FIG. 6 is a graph showing the elution profile of saccharide fragmentsgenerated from type III GBS capsular polysaccharide after treatment withozone.

FIG. 7A-7C are the ¹ H NMR spectra of type III GBS nativepolysaccharides (A), and ozonolysis-generated saccharide fragments ofthree (B) and two (C) repeating units.

FIG. 8A-8D are graphs showing the elution profiles of the saccharidefragments generated from type III GBS polysaccharide following treatmentwith ozone for 150, 195, 270, and 355 minutes, respectively.

FIG. 9A-9B are graphs showing the repeating unit structure ofpolysaccharide A of Bacteroides fragilis (9A), and the elution profilefollowing liquid chromatography of the saccharide fragments generatedfrom polysaccharide A of Bacteroides fragilis (9B).

FIG. 10 is a graph showing the molecular weight of saccharide fragmentsafter treatment of a starting type III CBS polysaccharide with ozone forthe indicated lengths of time.

FIG. 11 is the ¹ H NMR spectrum of type III GBS native polysaccharidesfollowing ozonolysis in NaHCO₃ buffer.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides new methods for cleaving polysaccharides usingozonolysis. In one method, ozonolysis is carried out in three steps: 1)hydroxyl groups on the polysaccharide are protected; 2) ozone is appliedto the protected polysaccharide to form a partially oxidizedintermediate containing an aldonic acid ester; and 3) the aldonic acidester intermediate is deprotected and hydrolyzed, thereby cleaving thestarting polysaccharide into saccharide fragments.

In a second method, ozonolysis is carried out in one step: ozone isapplied directly to a polysaccharide, which can contain saccharidesubunits joined in either α or β linkages, in an aqueous solution. Whenthe polysaccharides are joined in a β-D glycosidic linkage, ozonolysismay generate an ester or a lactone as described above.

Any polysaccharide is generally a suitable starting material for theozonolysis methods. Polysaccharides can be purchased commercially orisolated from natural sources by standard methods. For example,polysaccharides can be isolated from bacterial species by methodsdescribed by Wessels et al., J. Biol. Chem. 266:6714 (1991), Tzianaboset al., J. Biol. Chem. 267:18230 (1992), and Lee et al., Infect. Immun.61:1853 (1993).

Ozonolysis Using the Three-Step Method

This method can be used to selectively depolymerize polysaccharidescontaining a β-D or α-L glycosidic linkage. The first step of the methodprotects the free hydroxyl groups of the polysaccharide from subsequenttreatment with ozone. Among the available protection methods,peracetylation is generally preferred, although other methods such aspersilylation and permethylation are also suitable. Peracetylation isusually accomplished by treating polysaccharides with acetic anhydrideand pyridine; however, acetic anhydride/potassium acetate or acetateanhydride/sodium acetate and the like can also be used as acetylationreagents.

If the polysaccharide is not soluble in acetic anhydride/pyridine, acosolvent such as formamide may be added. The reaction time can beshortened by increasing the temperature. For example, the reaction takesplace either overnight (in 12-24 hours) at room temperature, or in 2hours at 70° C.

Upon completion of the peracetylation reaction, the excess reagents andsolvents are removed using procedures known in the art. For example, thereaction mixture can be dialyzed against distilled water, after whichthe water is removed by lyophilization or evaporated under nitrogen oron a rotary evaporator. Alternatively, the solvent can be directlyremoved using a rotary evaporator. Direct removal typically requiresheating or the addition of ethanol to speed the evaporation of pyridineand formamide.

In the next step, the protected polysaccharide is dissolved in ethylacetate, acetic anhydride/potassium acetate, or another ozone-inertsolvent such as dichloromethane or tetrahydrofuran. The solution issonicated for a few minutes to dissolve the polysaccharide, after whichozone is added at room temperature. To reduce the amount of solventevaporated, a condensation device can be used during the ozoneapplication step.

The application of ozone to the protected polysaccharide results in theformation of an aldonic acid ester intermediate. Various methods ofapplying the ozone may be used. For example, ozone can be delivered froman external ozone generator (More-Zon10, More Production, Taiwan), whichcreates ozone electronically from oxygen or air. Other ozone applicationmethods may also be used. After ozone treatment, the solvent isevaporated on a rotary evaporator.

In the third step, the aldonic ester linkages in the polysaccharide canbe hydrolyzed with a base such as 0.1 N NaOH at room temperature for 30minutes, which simultaneously removes the protecting group. The nascentoligomers can alternatively be liberated, and the termini simultaneouslyfunctionalized, with another nucleophile known to cleave ester bonds.Appropriate nucleophiles include (but are not limited to) alkoxides,phenoxides, carbanions, thiols, and hydrazines. The use of anα,ω-diamine, for example, leads to an amide linkage of the saccharidesto one end of the amine, with the free amino group available forcoupling to a carrier or support matrix.

Alternatively, the aldonic esters may be converted to lactones by simpleheating, and the acetyl protecting groups may then be removed in aseparate subsequent step.

Ozonolysis Using the One-Step Method

In the one-step method, degradation of the polysaccharide isaccomplished in one step by treating the polysaccharide solution withozone. The polysaccharide substrates are dissolved in any suitableaqueous solvent or buffer solution, e.g., water. For degradingpolysaccharides containing acid-sensitive residues, the reaction ispreferably carried out in a basic buffer (e.g., phosphate bufferedsaline, or sodium bicarbonate) to prevent the loss of acid-sensitivegroups. Acid formed during the ozonolysis reaction may also beneutralized with a base such as alkali, alkali carbonates, bicarbonates,hydroxides, or other inorganic or organic bases.

Polysaccharides containing either α or β linkages, including α-L, α-D,β-L or β-D linkages, are suitable starting products for the one-stepozonolysis method. When the polysaccharide contains a β-D or α-Llinkage, it forms a partially oxidized intermediate containing analdonic acid ester, which forms a lactone and automatically cleaves thepolysaccharide. Ozone treatment will preferentially affect the β-D orα-L linkages; thus, in relatively brief exposures to ozone,polysaccharides containing β-D or α-L linkages can be preferentiallydepolymerized at these sites.

When polysaccharides are exposed to ozone for lengthy periods of time inan aqueous solution, additional reactions can occur that can result,e.g., the formation of radicals, in cleavage of glycosidic bonds in thepolysaccharide, oxidation of the polysaccharides, or the formation ofacids. Because these reactions do not require the presence of a β-D orα-L glycosidic linkage, they can be used to cleave polysaccharidescontaining only α linkages (e.g., dextran or starch). By monitoring theextent of ozonolysis, e.g., by monitoring products subjected toozonolysis for varying lengths of time, the desired reaction productscan be obtained.

Further Processing of Saccharide Fragments Generated Using Ozonolysis

The products resulting from the ozonolysis methods are saccharidefragments terminating with a carboxylate group. The carboxylate groupcan be activated with carbodiimides that function as zero-lengthcross-linking agents and couple the saccharides to amine-containingmolecules.

All the resulting saccharide fragments may be further manipulated forother purposes. Saccharide fragments that contain one or more diolfunction groups may be selectively oxidized with sodium metaperiodate tocreate aldehyde groups. For example, sialic acid can easily be oxidizedby sodium periodate to create a free aldehyde group at the C8 position.Such saccharide fragments may then be coupled to molecules containingamine moieties, such as proteins, or to a bifunctional molecule thatserves as a spacer and can be further coupled to another molecule.

For polysaccharides containing β-D linkages, the ozonolysis-mediatedcleavage is highly selective in that ozone reacts selectively at theselinkages; however, the cleavage site is generally random among all thesame β-D glycosidic linkages within a polysaccharide. The sizedistribution of saccharides may be controlled by controlling ozonolysisconditions, including the concentration of the polysaccharide, thereaction time, the rate of ozone passing through the reaction mixture,and the total amount of ozone consumed. For example, longer reactiontimes and consumption of more ozone result in smaller saccharides.Controlled cleavage of the polysaccharide thus results in a mixture ofsaccharides with a desired, narrow range of sizes which retain therepeating-unit structures of the parent polysaccharide.

The products of the ozonolysis reaction can be separated by techniqueswell known in the art, e.g., gel-filtration, size-exclusion, orion-exchange column chromatography. The eluent can be a suitable buffer,such as PBS, TRIS, or distilled water. Fractions are monitored by arefractive index detector or are assayed for carbohydrate contents.Fractions representing different-sized saccharides are pooled andanalyzed by spectroscopic methods, typically NMR spectroscopy. The sizesof the resulting saccharide fragments are determined either by massspectrometry (e.g., electrospray) or by measurement of their elutionvolume and calculation from the calibration curve of the column.Maintenance of polysaccharide function can be verified by an appropriateassay (e.g., an ELISA).

An important class of polysaccharides suitable for use in the inventioninclude bacterial capsular polysaccharides and lipopolysaccharides,e.g., those from the pathogenic bacteria group B Streptococcus, B.fragilis, S. aureus, and S. pneumoniae. Protective polysaccharidesassociated with these bacteria contain labile sialic acid or pyridyl(carboxyethylidene) residues that are critical to protective epitopes.

The saccharide fragments generated from these polysaccharides byozonolysis can be used as diagnostic reagents, therapeutic reagents, oras reagents for the preparation of vaccines. In these applications,fragments of the outer polysaccharide coats of the organisms can beincorporated into a molecular matrix or attached to a carrier.

The following non-limiting examples are used to describe the generationof saccharide fragments by ozonolysis and uses of the saccharidefragments so generated.

EXAMPLES

Materials and Methods

Unless otherwise indicated, the following materials and methods wereused in performing the experiments described in the following examples.

Group B Streptococcus capsular polysaccharides were isolated andpurified as described by Wessels et al., J. Biol. Chem. 266:6714 (1991).Preparation of polysaccharide A of Bacteroides fragilis was as describedby Tzianabos et al., J. Biol. Chem. 267:18230 (1992), and thepreparation of the capsular polysaccharide of Staphylococcus aureus type5 was as described by Lee et al., Infect. Immun. 61:1853 (1993).Desialyated GBS has a structure identical to the polysaccharide ofStreptococcus pneumoniae type 14. It was obtained by mild acidhydrolysis to remove the sialic acid from the GBS polysaccharide asfollows: One 10 mg sample of GBS type III polysaccharide was added to 5ml of 6% acetic acid and heated at 80° C. for 1 hour. The sample wasthen dialyzed against deionized water and freeze-dried. Dextran waspurchased from Pharmacia (Piscataway, N.J.).

Superdex75 and Superose12 columns were obtained from Pharmacia LKBBiotechnology, Inc.

Peracetylation

A 10 mg sample of polysaccharide was dissolved in 5 ml of formamide andtreated with 1 ml of pyridine and 0.5 ml of acetic anhydride. Themixture was magnetically stirred at room temperature for 16 hrs, thendialyzed extensively against distilled water and freeze dried.

Ozone Treatment

A 10-ml volume of ethyl acetate was added to the dried product, and themixture was bubbled with 21% ozone at a flow rate of 3.17 ml/sec for 5hours unless indicated otherwise. Ozone was generated from compressedair or oxygen through an ozone generator (More-Zon10). The solvent wasthen removed by evaporation on a rotary evaporator.

For the one-step ozonolysis procedure, ozonolysis was carried out in anaqueous buffer. A 10 mg sample of polysaccharide was dissolved in 2 mlof water and bubbled for the indicated period of times. Polysaccharidescontaining acid-sensitive groups such as those from GBS and B. fragiliswere dissolved in either 0.2 M PBS ph 7.2 or 0.1 M NaHCO₃ pH 8.6.

Hydrolysis of the Ester Intermediate

In alkaline hydrolysis, the dried, oxidized material was mixed with 5 mlof 0.1N NaOH at room temperature for 2 hours and then neutralized withdilute acetic acid or hydrochloric acid. For allylamine hydrolysis, theoxidized product was treated with 5 ml of allylamine at room temperaturefor 30 min. The excess allylamine was evaporated under a stream ofnitrogen in a hood.

Separation of Saccharide Fragments

The saccharide mixture was separated with an FPLC system (Pharmacia)using a Superdex 75 or Superdex 30 column by eluting with 0.3 mM PBSbuffer with 0.025% azide at pH 7.2. Fractions were monitored by arefractive index detector. Those in desired size ranges were pooled andanalyzed by spectroscopic methods. The columns were calibrated withdextran standards, and the molecular weights of saccharide fragmentswere obtained from column calibration curves.

In experiments using the one-step ozonolysis method, the sizes ofsaccharides were obtained using a Superose 12 column that had beencalibrated with a dextran standard. The sizes of the saccharides werecalculated from their molecular size versus their elution volumefunction. The structures of the polysaccharides were analyzed by ¹ H NMRspectroscopy.

Instrumental Methods

NMR analyses were performed on a Varian VXR500 spectrometer (Palo Alto,Calif.) or a Bruker AMX 500 with a proton resonance frequency of 500MHz. ¹ H spectra were recorded at 70° C. in D₂ O. Proton chemical shiftswere referenced relative to water resonance calibrated at 4.290 ppm at70° C., 4.632 ppm at 37° C., and 4.755 ppm at 25° C.

Example 1

Generation of Saccharide Fragments from Type II GBS PolysaccharidesUsing the Three-Step Ozonolysis Procedure

Type II GBS polysaccharide (FIG. 1) was prepared as described andtreated with ozone for five hours. The saccharide fragments wereseparated on a Superdex 75 column (HiLoad™16/60, prep grade, Pharmacia),which has a size separation range of 0.5-30 kDa. The fractions weremonitored by a differential refractometer (WATERSR401™, Millipore Corp.,Bedford, Mass.).

The eluted fractions are shown in FIG. 2. Three peaks were detected anddesignated 1, 2, and 3. Based on the column calibration curve, peaks 1and 2 had average molecular weights of 2.7 kDa and 4.3 kDa,respectively. As the native type II GBS polysaccharide has aheptasaccharide repeating unit of 1.3 kDa, these peaks corresponded totwo and three repeating units, respectively. At peak 3 and the highermolecular weight peaks, saccharide fragments with four and morerepeating units elute.

The ¹ H NMR spectra of the type II GBS native polysaccharide and thesaccharide fragments of peaks 1-3 are shown in FIG. 3. The NMR structureof the native polysaccharide reveals a heptasaccharide repeating unit,as indicated by the six anomeric protons between 4.4 ppm and 5.2 ppmfrom the hexose residues, along with a sialic acid residue, as revealedby its ¹ H-resonance at 2.85 ppm and 1.86 ppm (FIG. 3A).

FIG. 3B shows that the majority of peak 3 is a saccharide fragment offour repeating units with an NMR spectrum identical to that of thenative polysaccharide. Similarly, FIGS. 3C and 3D demonstrate that peaks2 and 1 correspond to saccharides of three and two repeating units,respectively. In FIG. 3D, the 11 anomeric signals at 4.90 ppm (2), 4.84ppm (2), 4.76 ppm, 4.66 ppm, 4.62 ppm, and 4.50 ppm (4) correspond tothe 11 hexose residues, along with two sialic acid residues and aterminal aldonic residue, of the two repeating units. The sialic acidresidue is retained in all fragments, as indicated by its ¹ H signals at2.85 ppm and 1.86 ppm in all the spectra.

Example 2

Generation of Saccharide Fragments from Type VIII GBS PolysaccharidesUsing the Three-Step Ozonolysis Procedure Following Ozonolysis forVarying Amounts of Time

The LC profile of type VIII GBS polysaccharide exposed to ozone for 20,40, or 80 hours is shown in FIG. 4. The saccharide fragments wereseparated on a Superdex 75 column (separation range, 0.5-30 kDa), with0.3 mM PBS as the elution buffer. As the reaction time increased, theaverage size of the saccharide fragment decreased. After 40 hours, theaverage size of the saccharides was 15 kDa. After 80 hours, the majorproduct corresponded to a tetrasaccharide repeating unit with amolecular weight of 803.

The ¹ H NMR spectrum of pooled fragments with average molecular weightsof 7 kDa is shown in FIG. 5. There are three anomeric proton resonancesbetween 4.8 and 5.0 ppm from the glucose, galactose, and rhamnoseresidues, respectively. The doublet at 1.4 ppm is due to the 6-deoxyprotons of the rhamnose residue. The signals at 2.9 ppm and 1.9 ppm aredue to the 3-H of the sialic acid residue. The spectrum of the pooled7-kDa fractions is identical to that of the native polysaccharide,indicating that the saccharide retained the parental repeat structure.

Example 3

Generation of Saccharide Fragments from Type III GBS PolysaccharidesUsing the Three-Step Ozonolysis Procedure

The LC profile of saccharide fragments generated upon treatment of typeIII GBS polysaccharides with ozone is shown in FIG. 6. Saccharidefragments were separated on a Superdex 75 column and eluted with 0.3 mMPBS. Peaks 1 and 2 correspond to saccharide fragments containing two andthree copies of the type III repeats, respectively.

FIG. 7 shows the ¹ H NMR spectra of the native type III GBSpolysaccharide and of peaks 2 and 1 of FIG. 6, respectively. The nativepolysaccharide (FIG. 7A) has a pentasaccharide repeating unit, asrevealed by the four anomeric protons (between 4.4 ppm and 4.8 ppm) fromthe four hexose residues, and a sialic acid residue, as revealed by its3-H resonance at 2.77 ppm and 1.80 ppm. Peak 2 (FIG. 7B) is mainly asaccharide of three repeating units. Its NMR spectrum is identical tothat of the native polysaccharide. Peak 1 (FIG. 7C) corresponds to asaccharide of two repeating units (10 residues). There are seven hexoseresidues as shown by the 7 anomeric signals at 4.78 ppm (1), 4.63 ppm(2), 4.55 ppm (2), and 4.48 ppm (2). In addition, there are two sialicacid residues and a terminal aldonic acid residue. The sialic acidresidue is retained in all fragments, as indicated by its 3-H signals at2.77 ppm and 1.80 ppm in all the spectra.

The effect of ozonolysis lasting for various periods on type IIIpolysaccharides was examined. FIG. 8 shows the LC profile afterozonolysis for 150, 195, 270, and 355 minutes, respectively. At eachtime point, the saccharides generated were of uniform size and fellwithin narrow size distributions.

The sizes of the saccharide fragment products for each duration ofozonolysis were determined with a Superose 12 column, which has a sizeseparation range of 1-300 kDa. The average sizes of the saccharides atthe time points shown in FIGS. 8A through 8D corresponded to 42, 23, 21,and 20 kDa, respectively. Upon prolonged reaction times of 455, 625, and820 minutes, the average sizes of the saccharides obtained were 16, 14,and 5 kDa, respectively (data not shown).

Example 4

Generation of Saccharide Fragments from Polysaccharide A of Bacteroidesfragilis using the Three-Step Ozonolysis Procedure

The repeating unit of the polysaccharide A from Bacteroides fragilis hasthe structure shown in FIG. 9A. FIG. 9B shows the LC profile of thesaccharides generated upon ozonolysis treatment of the Bacteroidesfragilis polysaccharide A using a Superdex75 column. The averagemolecular weights for peaks 1-3 were 2.1, 4.3, and 6.6 kDa,respectively.

Example 5

Preparation of Tetanus Toxoid Conjugate Vaccine Using SaccharideFragments Produced by the Three-Step Ozonolysis Procedure

A saccharide fragment from a type III GBS polysaccharide (5 mg) obtainedafter ozonolysis was dissolved in 0.375 ml of water and oxidized with0.125 ml of 0.01 M sodium metaperiodate at room temperature in the darkfor 90 minutes (Wessels et al., J. Clin. Invest. 86:1428 (1990)). Themixture was then dialyzed against water and lyophilized. The oxidizedsaccharide sample was combined with 4 mg of tetanus toxoid, and thecombination was dissolved in 0.3 ml of 0.1 M NaHCO₃ (pH 8.2), with 20 mgof sodium cyanoborohydride added. The mixture was incubated at 37° C.overnight. The conjugate vaccine product was purified on a S-300 column(Pharmacia).

Example 6

Generation of Saccharide Fragments from β-Containing Polysaccharides andα-Containing Polysaccharides Using the One-Step Ozonolysis Procedure

To determine if ozonolysis could be performed using the one-stepprocedure, polysaccharides of GBS type III, type 14 S. pneumoniae, ordextran were used as the starting material.

A sample of GBS type III polysaccharide (8.3 mg) was dissolved in 1 mlof water and bubbled with ozone for 47 minutes. During this time the pHof the solution was monitored. The reaction mixture became slightlyacidic after 20 minutes of ozonolysis, at which time a few drops of a0.033 M NaHCO₃ solution was added until the solution returned to neutralpH. At various time intervals, a 30 μl aliquot of the reaction mixturewas taken and screened on a Superose 12 column to check the size of theproducts.

As is shown in FIG. 10, the size of the products decreased vary rapidly.After 32 minutes, the reaction again became acidic, and 0.1N NaOH wasadded until the pH of the solution reached 9. The reaction was continuedfor another 15 minutes, during which time the pH of the solutionremained unchanged. The average molecular weight of the final saccharidefragment was 4.4 kDa. The product was purified on a P2 Biogel column(Biorad, Hercules, Calif.) and eluted with water on a FPLC system. Onemajor peak was obtained.

The collected fractions from the FPLC column were then pooled,lyophilized, and subjected to ¹ H NMR spectroscopy. The ¹ H NMR spectrumis shown in FIG. 11. The spectra was identical to that of the startingpolysaccharide, thus demonstrating that the saccharide fragment producthas the same subunit structure as the starting polysaccharide. Inparticular, the acid-labile sialic residue of the type III GBS wasretained as shown by the characteristic H-3 resonances at 2.8 and 1.8ppm. These data suggest that ozonolysis using the one-step procedureresults in saccharide fragments having the same internal repeatstructure as the starting polysaccharide.

The one-step ozonolysis method was also carried out using the type 14 S.pneumoniae polysaccharide as the starting substrate. A 10.5 mg samplewas dissolved in 5 ml of 0.033 M NaHCO₃ solution and ozonized for 5hours. During the ozonolysis 75 μl aliquots of the reaction mixture weretaken and screened on a Superose 12 sizing column. The nativepolysaccharide has an average molecular weight of 130 kDa. After thereaction had proceeded for 30, 140, and 330 minutes, the size of thesaccharides in the reaction mixture decreased to 23, 6, and 3 kDa,respectively. ¹ H NMR analyses of the saccharide fragments revealed thatthe internal structure of the polysaccharide was conserved in thesefragments.

The results using type III GBS polysaccharide and type 14 S. pneumoniaepolysaccharide demonstrate that polysaccharides containing β linkagescan be cleaved to saccharide fragments while retaining the subunitlinkages. To determine if saccharide fragments could also be producedfrom a polysaccharide containing α linkages, the one-step ozonolysisprocedure was carried out using dextran as the starting polysaccharide.Dextran is a polysaccharide composed exclusively of the D-glucopyranosylunits connected via an α-(1,6) linkage.

A 4.9 mg sample of dextran (molecular weight of 110 kDa) was dissolvedin 2.4 ml of 0.03 M NaHCO₃ and subjected to ozonolysis for 2.5 hours,with periodic monitoring of the pH of the solution. The solutiongradually became acidic, and at the termination of the ozonolysisreached a pH of 1. The size of the reaction products was monitored andfound to be 300 Da. These results demonstrate that polysaccharidescontaining α-linkages can also be cleaved using ozonolysis.

In addition to the above-described application of the one-stepozonolysis procedure to polysaccharides derived from GBS type III and S.pneumoniae type 14, this method has also been used successfully toproduce saccharide fragments from B. fragilis type A, cellobiose, andlactose.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for producing a saccharide fragmentproduct by degrading a larger polysaccharide, the polysaccharide beinglarger than the saccharide fragment product and comprising at least onecovalent bond between a C1 anomeric carbon of an aldose residue and anoxygen atom of a second residue in a β-D or α-L glycosidic linkage; themethod comprising:a) protecting free hydroxyl groups on the largerpolysaccharide; b) reacting the larger polysaccharide with ozone tooxidize the C1 anomeric carbon, thus converting the aldose residue to analdonic acid ester residue; and c) cleaving the aldonic acid esterresidue to form the saccharide fragment.
 2. The method of claim 1wherein said polysaccharide contains sialic acid.
 3. The method of claim1 wherein said polysaccharide is a group B Streptococcus capsularpolysaccharide.
 4. The method of claim 1 wherein said polysaccharide isthe O-antigen of a lipopolysaccharide.
 5. The method of claim 1 whereinsaid polysaccharide is a capsular polysaccharide of Staphylococcusaureus.
 6. The method of claim 5 wherein said polysaccharide is thecapsular polysaccharide of Staphylococcus aureus type 5 orStaphylococcus aureus type
 8. 7. The method of claim 1 wherein saidpolysaccharide is the capsular polysaccharide of Streptococcuspneumonia.
 8. The method of claim 1 wherein said polysaccharide is thecapsular polysaccharide of Bacteroides fragilis.
 9. The method of claim1 wherein said polysaccharide is selected from the group consisting ofGBS type II polysaccharide, GBS type III polysaccharide, Bacteroidesfragilis capsular polysaccharide, and GBS type VIII polysaccharide. 10.The method of claim 1 wherein the ozone is added as a solution.
 11. Themethod of claim 1 wherein the ozone is generated in-situ.
 12. The methodof claim 1 wherein the ozone is delivered from an external source. 13.The method of claim 1 wherein the ester is cleaved by a nucleophile. 14.The method of claim 13 wherein the nucleophile is a hydroxyl ion. 15.The method of claim 13 wherein the nucleophile is an amine.
 16. Themethod of claim 13 wherein the nucleophile is a thiol.
 17. The method ofclaim 13 wherein the nucleophile is a carbanion.
 18. The method of claim1 wherein the ester is cleaved by heating.
 19. The method of claim 1wherein the hydroxyl groups are protected by forming ester groups.
 20. Amethod for producing a saccharide fragment product by oxidizing a largerpolysaccharide, the polysaccharide being larger than the saccharidefragment product and comprising at least one covalent bond between a C1anomeric carbon of an aldose residue and an oxygen atom of a secondresidue in a glycosidic linkage; the method comprising reacting thelarger polysaccharide with ozone to yield a mixture comprising thesaccharide fragment, wherein said reaction takes place in an aqueoussolution.
 21. The method of claim 20 wherein said covalent bond betweenthe C1 anomeric carbon of the aldose residue and the oxygen atom of thesecond residue is in an α glycosidic linkage.
 22. The method of claim 20wherein said covalent bond between the C1 anomeric carbon of the aldoseresidue and the oxygen atom of the second residue is in an α glycosidiclinkage.
 23. The method of claim 22 wherein said larger polysaccharidereacts with ozone to oxidize the C1 anomeric carbon, thus converting thealdose residue to an aldonic acid ester residue.
 24. The method of claim22 wherein said polysaccharide is a group B Streptococcus capsularpolysaccharide.
 25. The method of claim 22 wherein said polysaccharideis the O-antigen of a lipopolysaccharide.
 26. The method of claim 22wherein said polysaccharide is a capsular polysaccharide ofStaphylococcus aureus.
 27. The method of claim 26 wherein saidpolysaccharide is the capsular polysaccharide of Staphylococcus aureustype 5 or Staphylococcus aureus type
 8. 28. The method of claim 20wherein said polysaccharide is the capsular polysaccharide ofStreptococcus pneumonia.
 29. The method of claim 20 wherein saidpolysaccharide is the capsular polysaccharide of Bacteroides fragilis.30. The method of claim 20 wherein said polysaccharide is selected fromthe group consisting of GBS type II polysaccharide, GBS type IIIpolysaccharide, Bacteroides fragilis capsular polysaccharide, and GBStype VIII polysaccharide.
 31. The method of claim 20 wherein the ozoneis added as a solution.
 32. The method of claim 20 wherein the ozone isgenerated in-situ.
 33. The method of claim 20 wherein the ozone isdelivered from an external source.
 34. The method of claim 23 whereinthe ester is cleaved by a nucleophile.
 35. The method of claim 34wherein the nucleophile is a hydroxyl ion.
 36. The method of claim 34wherein the nucleophile is an amine.
 37. The method of claim 34 whereinthe nucleophile is a thiol.
 38. The method of claim 34 wherein thenucleophile is a carbanion.
 39. The method of claim 23 wherein the esteris cleaved by heating.